Donate Help Contact The AHA Sign In Home
American Heart Association
Stroke
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Stroke. 2002;33:1093-1100
doi: 10.1161/01.STR.0000014205.05597.45
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sommer, C.
Right arrow Articles by Kiessling, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sommer, C.
Right arrow Articles by Kiessling, M.
Related Collections
Right arrow Gene expression
Right arrow Gene regulation
Right arrow Genomics
Right arrow Ischemic biology - basic studies

(Stroke. 2002;33:1093.)
© 2002 American Heart Association, Inc.

Original Contributions

Ischemia and Ischemic Tolerance Induction Differentially Regulate Protein Expression of GluR1, GluR2, and AMPA Receptor Binding Protein in the Gerbil Hippocampus

GluR2 (GluR-B) Reduction Does Not Predict Neuronal Death

Clemens Sommer, MD Marika Kiessling, MD

From the Department of Neuropathology, Ruprecht-Karls University of Heidelberg (Germany).

Correspondence to Dr Clemens Sommer, Laboratory of Neuropathology, Department of Pathology, University of Ulm, Albert-Einstein-Allee 11, D-89081, Ulm, Germany. E-mail clemens.sommer{at}medizin.uni-ulm.de


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose— Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons has been suggested to occur as a result of formation of calcium-permeable {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors lacking the GluR2 subunit (GluR2 hypothesis). DND can be prevented by a short tolerance-inducing ischemic period. The present study was designed to assess whether postischemic protein levels of GluR2 predict neuronal death. Additionally, the role of AMPA receptor binding protein (ABP) was investigated with respect to neuronal death or survival.

Methods— Postischemic protein expression of GluR1, GluR2, and ABP was analyzed in 3 experimental paradigms of transient global ischemia with the use of subunit-specific antisera and semiquantitative densitometric evaluation. Gerbils were subjected (1) to a 5-minute ischemic period resulting in DND of CA1 neurons; (2) to a 2.5-minute period of ischemia mediating tolerance induction; and (3) to 5 minutes of ischemia in the ischemia-tolerant state (2.5+5 minutes of ischemia 4 days apart).

Results— The major finding was that GluR2 protein levels were significantly downregulated in neuronal subpopulations destined to survive, ie, in CA1 principal neurons after ischemic tolerance induction and in the ischemia-tolerant state, as well as in CA3 neurons after a 5-minute period of ischemia. ABP expression remained unaffected.

Conclusions— Our results modify the GluR2 hypothesis in that postischemic GluR2 reduction also occurs in hippocampal CA1 and CA3 principal neurons without subsequent neuronal death. ABP is obviously not involved in mechanisms of DND or ischemic tolerance induction.


Key Words: brain ischemia • immunohistochemistry • ischemic preconditioning • receptors, AMPA • gerbils


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Transient cerebral forebrain ischemia of only 5 minutes’ duration causes selective and delayed neuronal death (DND) of hippocampal CA1 neurons in the gerbil brain.1 These highly vulnerable CA1 neurons can be protected against irreversible ischemic damage by a preconditioning stimulus, eg, a short sublethal period of ischemia.2–4 The precise molecular mechanisms of both phenomena, DND and endogenous neuroprotection of CA1, however, are still under discussion. An increase of cytosolic free calcium caused by neurotoxic activation of receptor- or voltage-operated ion channels is thought to play a major role in DND.5 Recent experimental evidence points to an additional or alternative zinc neurotoxicity after cerebral ischemia.6 Since pharmacological blockade of calcium-permeable N-methyl-D-aspartate (NMDA) receptors does not prevent postischemic degeneration of CA1 neurons,7,8 interest has focused increasingly on {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, another major subtype of glutamate receptors. Recent evidence suggests that AMPA receptors are tetramers that can be assembled from 4 different subunits, GluR1 to GluR4 (GluR-A to GluR-D).9–13 They are impermeable to calcium ions as long as at least 1 GluR2 subunit in its edited form is present.14–16 Previous work demonstrated that GluR2 editing is not altered by global ischemia and is unlikely to contribute to neuronal degeneration.17,18 The observation that 1 GluR2 subunit can determine the calcium permeability of AMPA receptors in conjunction with a postischemic downregulation of GluR2 mRNA levels at 24 hours led to the so-called GluR2 hypothesis, suggesting a contribution of calcium-permeable AMPA receptors to DND.19–21

AMPA receptor binding protein (ABP) is a recently cloned novel component of the postsynaptic density22 that binds to the C termini of GluR2 and GluR3 subunits via PDZ domains and is involved in the regulation of AMPA receptor function by dynamically regulating postsynaptic localization and clustering of AMPA receptors.23,24 To address the question of whether ABP is also involved in mechanisms of DND or ischemic tolerance induction, interest in the present study also focused on a possible link between the kinetics of GluR2 protein expression and changes in ABP expression.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animal Experiments
Experiments were performed on adult male Mongolian gerbils (Meriones unguiculatus; weight, 70 to 80 g) obtained from Charles River Deutschland (Sulzfeld, Germany). Animals had free access to food and water before experiments. Gerbils were subjected to transient forebrain ischemia by bilateral occlusion of the common carotid artery. Ischemic tolerance was induced according to the protocol of Kirino et al4 with minor modifications.25 Anesthesia was achieved with a mixture of 30% O2, 70% N2O, and 1.5% halothane. Three experimental groups of animals were investigated. One group was subjected to a single 5-minute period of ischemia, resulting in DND of CA1 neurons. The second group was subjected to a single tolerance-inducing 2.5-minute period of ischemia, and a third group was subjected to 5 minutes of ischemia in the ischemia-tolerant state (2.5+5 minutes of ischemia 4 days apart, a paradigm that results in survival of almost all CA1 neurons). Sham-operated control gerbils were subjected to anesthesia and all surgical procedures, except clamping of the carotid arteries. Animals were killed by transcardiac perfusion under deep ether anesthesia. Heparin (90 IU) was given via the left ventricle before washout of blood vessels with isotonic saline. Brains were perfusion-fixed with 4% (wt/vol) paraformaldehyde solution, removed, and postfixed overnight in the same fixative before vibratome sectioning. Postischemic expression of GluR1 and GluR2 subunit protein was investigated in sham-operated gerbils and in all experimental groups after recirculation intervals of 8, 24, 48, and 96 hours (n=4 to 9). All procedures were performed according to the guidelines of the German animal protection law.

Neuropathological Evaluation
Neuronal cell densities within the hippocampal pyramidal layers were quantitatively assessed after labeling neurons with an antibody against the neuronal marker protein NeuN as previously described.26,27 Six groups of gerbils were investigated at 96 hours after sham procedure or reperfusion: untreated control gerbils (n=4), gerbils subjected to a single (n=5) or repetitive (n=3) sham operation, and gerbils subjected to 2.5 minutes (n=8), 2.5+5 minutes (n=4), or 5 minutes (n=6) of ischemia. Hippocampal subfields CA1 and CA3 of both hemispheres were scanned at a magnification of x300. Two sets of 3 adjacent regions per area (strictly localized within the pyramidal cell layer of CA1 or CA3) per hemisphere were recorded, averaged, and expressed as mean cell number per square millimeter (neuronal density). All data were statistically analyzed with the use of the general statistics module of Analyze-it for Microsoft Excel (Analyze-it Software, Ltd). Values were expressed as mean±SEM. Significant group effects were confirmed by ANOVA and Bonferroni error protection with a significance level at P<0.05.

Immunohistochemistry
Immunohistochemistry was performed with antibodies against GluR1 protein (obtained from Chemicon International Inc), GluR2 protein (kindly provided by R. Wenthold), and ABP (kindly provided by E.B. Ziff), which were generated in rabbits immunized with bacterially expressed fusion proteins.22,28–30 All antibodies used are well characterized, and the specificity of antisera has been previously demonstrated.22,28–30 Immunohistochemistry was performed on coronal free-floating 50-µm vibratome sections. After pretreatment with methanol 70%/1% H2O2 for 20 minutes, free aldehyde moieties were blocked by a 10 minutes of incubation in PBS/0.4% borohydride. Then sections were incubated in normal swine serum (10% in PBS) for 30 minutes, followed by the primary antisera for 72 hours at 4°C. The primary antibodies were diluted 1:100 (GluR1), 1:200 (GluR2), and 1:250 (ABP). Then slices were washed with PBS and 0.2% Triton X-100, followed by an incubation period of 30 minutes at room temperature with a secondary biotinylated anti-rabbit IgG antibody. After a rinse with PBS and 0.2% Triton X-100, immunoreactivity was visualized by the avidin-biotin complex method (Vectastain, Vector Laboratories). Sections were developed in 0.02% diaminobenzidine with 0.02% hydrogen peroxide. The reaction product was intensified by addition of 0.02% cobalt chloride and nickel ammonium sulfate. Omitting the primary antisera in a subset of control slides resulted in no immunostaining (not shown).

For semiquantitative analysis of receptor protein expression, hippocampi were investigated by optical densitometry as previously described26 with minor modifications.31 All sections were examined at a final magnification of x40 with a Zeiss Axiophot microscope. Images were scanned in equal light conditions with a DMC video camera (Polaroid). From hippocampal subfields CA1 and CA3, 4 regions of interest (ROI) in the pyramidal layer (GluR1 and GluR2) or in dendritic layers (ABP), respectively, were selected on the monitor. ROIs were rectangular areas of 0.01 mm2 (0.05x0.2 mm). Optical density of immunoreactivity within the ROIs was automatically determined with AIS imaging research software (Imaging Research Inc). The optical density of the corpus callosum was used as reference value for background staining (ODUSP) and subtracted from total optical density in the ROI (ODTOT), resulting in specific optical density (ODSP). Specific immunolabeling was calculated as the ratio ODSP/ODTOT. Values (in percentages) are expressed as mean±SEM. Statistical analysis was performed with the use of the general statistics module of Analyze-it for Microsoft Excel (Analyze-it Software, Ltd). Significant group effects were confirmed by ANOVA and Bonferroni error protection with a significance level at P<0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Neuropathological Evaluation
Immunohistochemical analysis of NeuN-stained neurons in hippocampal subfields CA1 and CA3 revealed no significant differences between untreated control gerbils and gerbils subjected to a single or repetitive sham operation in both CA1 and CA3 (Table). A single 5-minute period of ischemia caused a significant reduction of neuronal cell density in the hippocampal CA1 subfield to 25% at 96 hours after reperfusion compared with sham-operated gerbils, whereas CA3 was unaffected (Table). Both a single tolerance-inducing 2.5-minute period of ischemia and a 5-minute period of ischemia in the ischemia-tolerant state led to focal neuronal loss in CA1 of some animals (not shown). This, however, did not result in a significant reduction of neuronal cell density in these experimental groups (Table). Again, CA3 remained unaffected (Table).


View this table:
[in this window]
[in a new window]
  		Table 1. Neuronal Cell Densities in the Ischemic Gerbil Hippocampus

Immunohistochemistry (GluR1, GluR2, and ABP)
Five Minutes of Ischemia
Prominent immunoreactivity for both GluR1 and GluR2 was present in the pyramidal layers of hippocampal subfields CA1 and CA3 in sham-operated gerbils (Figure 1). In CA1, GluR1 immunoreactivity was unchanged up to 48 hours of reperfusion and became restricted to a few scattered surviving interneurons at 96 hours (Figures 1 and 2). GluR1 immunoreactivity in CA3 remained unchanged at all time points investigated (Figures 1 and 2). Immunostaining for GluR2 in CA1 continuously decreased after ischemia, reaching significance at 48 and 96 hours. However, GluR2 immunoreactivity was not completely abolished 96 hours after reperfusion and yet was detectable in damaged shrunken CA1 neurons. In CA3, GluR2 immunoreactivity was significantly reduced between 8 and 48 hours, reattaining control levels at 96 hours after recirculation (Figures 1 and 2).



View larger version (128K):
[in this window]
[in a new window]
  		Figure 1. Hippocampal immunostaining for GluR1 (left), GluR2 (middle), and ABP (right) after a single 5-minute period of ischemia. In sham-operated control animals (Co), strongest immunoreactivity is present for GluR1 and GluR2 in the pyramidal layer of CA1 and CA3, whereas ABP immunoreactivity is accentuated in dendritic layers. Ninety-six hours after recirculation, GluR1 immunoreactivity in CA1 is restricted to scattered surviving interneurons but is unchanged in CA3. GluR2 immunoreactivity in CA1 continuously (see Figure 2) decreases after ischemia. Note that in CA3 neurons destined to survive, a marked decline of GluR2 immunoreactivity is evident at 8 and 48 hours, indicating that reduced GluR2 levels are not invariably associated with postischemic cell death. A reduction of ABP immunoreactivity is first detectable in CA1 at 96 hours after reperfusion in conjunction with neuronal death. Bar=500 µm.



View larger version (55K):
[in this window]
[in a new window]
  		Figure 2. Semiquantitative assessment of postischemic hippocampal GluR1, GluR2, and ABP immunoreactivity in various experimental paradigms. After a single 5-minute period of ischemia, GluR2 immunoreactivity is progressively reduced in CA1 compared with GluR1 and ABP but first reaches significance immediately before DND (a). In CA3, GluR2 immunoreactivity is significantly decreased between 8 and 48 hours but has nearly reattained control values at 96 hours (b). A single 2.5-minute tolerance-inducing ischemia significantly and selectively reduces GluR2 immunoreactivity in CA1 after 24 hours (c), whereas GluR1 and ABP are unaffected in both CA1 (c) and CA3 (d). Similarly, in the ischemia-tolerant state, GluR2 is transiently reduced at 8 hours (e), whereas GluR1 is nonsignificantly decreased and ABP remains unchanged in CA1 (e) and CA3 (f). OD-sp indicates specific optical density; OD-tot, total optical density. *Significant (P<0.05, ANOVA and Bonferroni error protection).

In sham-operated gerbils, ABP immunoreactivity in CA1 and CA3 was only weak in the pyramidal layer but was prominent in dendritic layers (Figure 1). Postischemic ABP immunoreactivity in CA1 was unaffected up to 48 hours after reperfusion but was significantly reduced at 96 hours (Figures 1 and 2). In CA3, no significant alterations of ABP protein expression were detectable (Figures 1 and 2).

Tolerance-Inducing Ischemia (2.5 Minutes)
Again, in sham-operated gerbils GluR1 immunoreactivity (not shown) and GluR2 immunoreactivity were prominent in neurons of the pyramidal layers of hippocampal subfields CA1 and CA3. A single 2.5-minute period of ischemia caused no alterations of the GluR1 immunoreactivity pattern in both CA1 and CA3 (Figure 2). GluR2 immunoreactivity in CA1, however, was transiently but significantly reduced at 24 hours (Figures 2 and 3). At 96 hours after reperfusion, GluR2 levels corresponded to those of sham-operated animals (Figures 2 and 3). Staining intensity of GluR2 immunoreactivity in CA3 was slightly but not significantly reduced between 24 and 96 hours (Figures 2 and 3).



View larger version (119K):
[in this window]
[in a new window]
  		Figure 3. Hippocampal immunostaining for postischemic GluR2 and ABP immunoreactivity after a tolerance-inducing 2.5-minute ischemic period. After tolerance induction, GluR2 immunoreactivity (left) in CA1 is transiently reduced at 24 hours. In CA3, a moderate but nonsignificant reduction is evident at 24 hours (see Figure 2). Note that ABP immunoreactivity (right) is unchanged at all time points investigated. Bar=500 µm.

ABP protein expression after a tolerance-inducing period of ischemia was not significantly altered in CA1 and CA3 at all time points investigated (Figures 2 and 3).

Five Minutes of Ischemia in the Ischemia-Tolerant State (2.5+5 Minutes of Ischemia 4 Days Apart)
In gerbils subjected to repetitive sham operation, the staining pattern and intensity for GluR1 and GluR2 corresponded to those of gerbils subjected to a single sham operation (not shown). GluR1 immunoreactivity in both CA1 and CA3 exhibited no significant alterations at all time points investigated (Figure 2). GluR2 immunoreactivity, however, showed a significant reduction at 8 hours in CA1 and was only slightly but nonsignificantly decreased at 8 and 24 hours in CA3 (Figures 2 and 4).



View larger version (121K):
[in this window]
[in a new window]
  		Figure 4. Hippocampal immunostaining for postischemic GluR2 and ABP immunoreactivity in the ischemia-tolerant state (2.5+5 minutes of ischemia). GluR2 immunoreactivity (left) in CA1 is markedly reduced at 8 hours of reperfusion. Note that in this and the previous ischemic paradigm (see Figure 3), severe GluR2 reduction is not followed by neuronal death (see Table). ABP immunoreactivity (right) is unchanged during the observation period, suggesting that GluR2 reduction occurs independently from its anchoring protein ABP. Co indicates control. Bar=500 µm.

As after the preconditioning ischemic period, ABP immunoreactivity in the ischemia-tolerant state was unchanged in CA1 and CA3 at all time points investigated (Figures 2 and 4).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Our study corroborates the GluR2 hypothesis in that, compared with GluR1, a 5-minute period of global ischemia severe enough to cause DND of CA1 pyramidal cells was associated with a continuous and selective decrease of GluR2 protein expression, which first reached significance at 48 hours after recirculation. This is a remarkable finding for several reasons. (1) In situ hybridization studies addressing postischemic GluR2 expression in CA1 yielded contradictory results: 4 studies showed a preferential decline of GluR2 mRNA in highly vulnerable CA1 neurons,19,20,32,33 whereas 3 other studies observed an unchanged ratio between the mRNAs encoding GluR1 and GluR2 subunits.34–36 (2) In contrast to global cerebral ischemia in rats with at least partially maintained postischemic protein synthesis in vulnerable CA1 neurons,37 total protein synthesis in the gerbil model of transient cerebral ischemia is almost completely and irreversibly inhibited in CA1.38,39 Despite this overall protein synthesis failure, however, heat shock protein 72 mRNA has been shown to be preferentially translated in postischemic gerbil hippocampal CA1 sector.40 Nevertheless, the translational deficit in CA1 neurons may have a profound impact on the respective protein expression pattern, and mRNA data alone do not predict postischemic abundance of a particular encoded protein. (3) On the protein level, Optiz and colleagues,41 using the same antibody as in the present study, first detected reduced postischemic GluR2 expression in CA1 neurons at 72 hours of reperfusion immunohistochemically and at 60 hours in microdissected CA1 sectors by immunoblotting. These data are somewhat at variance with our results. Ischemic damage of medial CA1 principal neurons precedes that of lateral pyramidal cells and frequently already manifests morphologically at 72 hours after reperfusion.42 At the same time point, nuclear DNA fragmentation is also detectable in the medial part of CA1.43 Thus, in agreement with these observations, 72 hours seems to be a critical time point beyond the threshold for incipient evidence of neuronal degeneration.

The validity of the GluR2 hypothesis has meanwhile been restricted by the original proponents to specific neuronal subpopulations. GluR2 knockdown experiments by antisense treatment led to a reduction of GluR2 in granule cells of the dentate gyrus but did not trigger granule cell degeneration, whereas neurons of CA1 and to a lesser extent neurons of CA3 underwent cell death,44 thus indicating a different sensitivity of neuronal subpopulations to GluR2 downregulation. Our present findings modify the GluR2 hypothesis in that diminished abundance of the GluR2 subunit in CA3 neurons after ischemia of 5 minutes’ duration (Figures 1 and 2) and even in the same neuronal subpopulation, ie, hippocampal CA1 neurons (at 24 hours after a single 2.5-minute period of ischemia and at 8 hours in the ischemic tolerant state), is not necessarily followed by cell death (Figures 2, 3, and 4). Similarly, Friedman and colleagues,36 using the rat model of global cerebral ischemia, recently demonstrated that postischemic hypothermia does not prevent reduction of GluR2 expression in CA1 seen in normothermic animals but results in survival of vulnerable CA1 neurons 7 days after reperfusion. Kjøller and Diemer45 analyzed postischemic GluR2 protein synthesis in the rat using Western blots and a pulse-labeling approach and also detected a significant decrease of the GluR2 synthesis rate at 24 hours in vulnerable CA1 and resistant CA3 and dentate gyrus neurons. These findings corroborate our data, strongly suggesting that significantly reduced GluR2 levels are not predictive of DND in general.

Further evidence against a direct causal relationship between lack of GluR2 and neuronal death comes from experiments with transgenic animals. The GluR2(-/-) mouse is viable and has a normal complement of CA1 and CA3 neurons. Moreover, there is no evidence of neuronal degeneration in the entire central nervous system, although calcium permeability of AMPA receptors is increased up to 9-fold.46 Mice with different levels of unedited GluR2 exhibit neurological abnormalities depending on the expression levels of the allele, but no direct neurotoxic effects are detectable.47,48 In addition, hippocampal GABAergic interneurons (physiologically lacking GluR2 subunits) also do not spontaneously degenerate.49,50 According to the GluR2 hypothesis, these neurons survive because they permanently express calcium-permeable AMPA receptors, whereas only neurons that suddenly have to cope with a high calcium influx through GluR2 lacking AMPA receptors undergo cell death. However, a recent study by Tóth and McBain51 demonstrated that interneurons of the stratum lucidum constitutively possess calcium-permeable AMPA receptors but still die after transient ischemia or kainate-induced seizures.52,53 In general, the notion that postischemic calcium influx through ion- or voltage-gated calcium channels with a concomitant increase of cytosolic free calcium inevitably results in irreversible cell damage has been reexamined: Recent in vitro work indicates that depletion of endoplasmic reticulum calcium stores and/or elevated calcium uptake in mitochondria may cause neuronal death, irrespective of free cytosolic calcium concentration.54–56

In regard to the regulation of AMPA receptor subunit expression after a preconditioning ischemic stimulus, to our knowledge there are only 3 studies, all from the laboratory of Diemer.35,45,57 Using in situ hybridization, these authors demonstrated that postischemic GluR2 expression in hippocampal CA1 neurons remains unchanged, whereas quantitative polymerase chain reaction analysis yielded a significant upregulation of GluR2 mRNA.35 The different PCR results are explained by polyadenylation of preexisting GluR2 mRNA, which in turn is thought to enhance synthesis of GluR2 protein,57 as reflected by a significant increase of GluR2 protein expression at 24 hours after preconditioning in microdissected CA1 regions assessed by immunoblotting.45 It cannot be excluded that the discrepancy with our immunohistochemical data, demonstrating reduced GluR2 protein expression in CA1 at 24 hours after tolerance induction (Figures 2 and 3), is due to an interspecies variation. More importantly, however, immunolabeling experiments by Western blotting do not distinguish between neuronal and nonneuronal GluR2 protein. In vitro, hippocampal astrocytes show GluR2 immunoreactivity,58 which may contribute to total GluR2 abundance in immunoblots. Moreover, homogenates do not reflect GluR2 subunit abundance of individual cells. To clarify this point, further experiments using in situ detection methods of GluR2 protein expression need to be performed in the preconditioned rat hippocampus.

In recent years, much effort was spent on the elucidation of the postsynaptic organization of excitatory synapses. A number of novel proteins of the postsynaptic density have been identified that interact with the C terminus of glutamate receptors, thereby controlling synaptic localization of these receptors. Two proteins have been demonstrated to interact specifically with GluR2/3 subunits of AMPA receptors: ABP22 and glutamate receptor interacting protein (GRIP).59 Since ABP and GRIP are essential for maintaining the synaptic surface accumulation of AMPA receptors,23 the persistence of normal ABP protein levels at different reperfusion intervals in different experimental paradigms of global ischemia despite reduced GluR2 levels indicates that a postischemic decrease of GluR2 expression is not related to a loss of its "anchoring" protein, ABP. The absence of changes in the postischemic expression of ABP, except in conjunction with DND, argues against a causal role of this protein for neuronal death or survival. Further investigations are necessary to pinpoint the mechanisms of GluR2 downregulation.

In conclusion, in agreement with the GluR2 hypothesis, our data demonstrate that CA1 neurons destined to die after global ischemia of 5 minutes’ duration exhibit reduced levels of GluR2 protein expression immediately before cell death. However, postischemic GluR2 protein levels were also substantially and significantly reduced in neuronal subpopulations destined to survive (in CA3 neurons after 5 minutes of ischemia and in CA1 neurons after induction of ischemic tolerance and in the ischemia-tolerant state), thus further restricting and modifying the GluR2 hypothesis. ABP, a novel AMPA receptor anchoring protein, appears to be unrelated to postischemic neuronal death or survival.


*    Acknowledgments
 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 317). The authors would like to thank Robert J. Wenthold (Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Md) and Edward B. Ziff (Howard Hughes Medical Institute, Department of Biochemistry, New York University Medical Center, New York, NY) for kindly providing the GluR2 and ABP antiserum, respectively. The authors acknowledge the excellent technical assistance of Stephan Hennes.

Received February 28, 2001; revision received August 1, 2001; accepted August 6, 2001.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982; 239: 57–69.[CrossRef][Medline] [Order article via Infotrieve]
  2. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, Kamada T, et al. "Ischemic tolerance" phenomenon detected in various brain regions. Brain Res. 1990; 561: 203–211.
  3. Kato H, Liu Y, Araki T, Kogure K. Temporal profile of effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res. 1991; 553: 238–242.[CrossRef][Medline] [Order article via Infotrieve]
  4. Kirino T, Tsujita Y, Tamura A. Induced tolerance to ischemia in gerbil hippocampal neurons. J Cereb Blood Flow Metab. 1991; 11: 299–307.[Medline] [Order article via Infotrieve]
  5. Choi DW. Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci. 1995; 18: 58–60.[CrossRef][Medline] [Order article via Infotrieve]
  6. Koh J-Y, Suh SW, Gwag BJ, He YY, Hsu CY, Choi DW. The role of zinc in selective neuronal death after transient global cerebral ischemia. Science. 1996; 272: 1013–1016.[Abstract]
  7. Buchan A, Li H, Pulsinelli W. The N-methyl-D-aspartate antagonist, MK-801, fails to protect against neuronal damage caused by transient, severe forebrain ischemia in adult rats. J Neurosci. 1991; 11: 1049–1056.[Abstract]
  8. Nellgard B, Wieloch T. Postischemic blockade of AMPA but not NMDA receptors mitigates neuronal damage in the rat brain following transient severe forebrain ischemia. J Cereb Blood Flow Metab. 1992; 12: 1–11.
  9. Keinänen K, Wisden W, Sommer B, Werner P, Herb A, Verdoorn TA, Sakmann B, Seeburg PH. A family of AMPA-selective glutamate receptors. Science. 1990; 249: 556–560.[Abstract/Free Full Text]
  10. Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science. 1992; 258: 597–603.[Abstract/Free Full Text]
  11. Mano I, Teichberg VI. A tetrameric subunit stoichiometry for a glutamate receptor-channel complex. Neuroreport. 1998; 9: 327–331.[Medline] [Order article via Infotrieve]
  12. Rosenmund C, Stern-Bach Y, Stevens CF. The tetrameric structure of a glutamate receptor channel. Science. 1998; 280: 1596–1599.[Abstract/Free Full Text]
  13. Chen GQ, Cui C, Mayer ML, Gouaux E. Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature. 1999; 402: 817–821.[CrossRef][Medline] [Order article via Infotrieve]
  14. Hollmann M, Hartley M, Heinemann S. Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition. Science. 1991; 252: 851–853.[Abstract/Free Full Text]
  15. Verdoorn TA, Burnashev N, Monyer H, Seeburg PH, Sakmann B. Structural determinants of ion flow through recombinant glutamate receptor channels. Science. 1991; 252: 1715–1718.[Abstract/Free Full Text]
  16. Washburn MS, Numberger M, Zhang S, Dingledine R. Differential dependence on GluR2 expression of three characteristic features of AMPA receptors. J Neurosci. 1997; 17: 9393–9406.[Abstract/Free Full Text]
  17. Paschen W, Schmitt J, Uto A. RNA editing of glutamate receptor subunits GluR2, GluR5, and GluR6 in transient cerebral ischemia in the rat. J Cereb Blood Flow Metab. 1996; 16: 548–556.[CrossRef][Medline] [Order article via Infotrieve]
  18. Rump A, Sommer C, Gass P, Bele S, Meissner D, Kiessling M. Editing of GluR2 RNA in the gerbil hippocampus after global cerebral ischemia. J Cereb Blood Flow Metab. 1996; 16: 1362–1365.[CrossRef][Medline] [Order article via Infotrieve]
  19. Pellegrini-Giampietro DE, Zukin RS, Bennett MVL, Cho S, Pulsinelli WA. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. Proc Natl Acad Sci U S A. 1992; 89: 10499–10503.[Abstract/Free Full Text]
  20. Gorter JA, Petrozzino JJ, Aronica EM, Rosenbaum DM, Opitz T, Bennett MVL, Connor JA, Zukin RS. Global ischemia induces downregulation of GluR2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil. J Neurosci. 1997; 17: 6179–6188.[Abstract/Free Full Text]
  21. Pellegrini-Giampietro DE, Gorter JA, Bennett MVL, Zukin RS. The GluR2 (GluR-B) hypothesis: Ca2+-permeable AMPA receptors in neurological disorders. Trends Neurosci. 1997; 20: 464–470.[CrossRef][Medline] [Order article via Infotrieve]
  22. Srivastava S, Osten P, Vilim FS, Khatri L, Inman G, States B, Daly C, DeSouza S, Abagyan R, Valtschanoff JG, Weinberg RJ, Ziff EB. Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP. Neuron. 1998; 21: 581–591.[CrossRef][Medline] [Order article via Infotrieve]
  23. Osten P, Khatri L, Perez JL, Köhr G, Giese G, Daly C, Schulz TW, Wensky A, Lee LM, Ziff EB. Mutagenesis reveals a role for ABP/GRIP binding to GluR in synaptic surface accumulation of the AMPA receptor. Neuron. 2000; 27: 313–325.[CrossRef][Medline] [Order article via Infotrieve]
  24. Scannevin RH, Huganir RL. Postsynaptic organization and regulation of excitatory synapses. Nat Rev Neurosci. 2000; 1: 133–141.[CrossRef][Medline] [Order article via Infotrieve]
  25. Sommer C, Gass P, Kiessling M. Selective c-JUN expression in CA1 neurons of the gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Brain Pathol. 1995; 5: 135–144.[Medline] [Order article via Infotrieve]
  26. Blümcke I, Becker AJ, Klein C, Scheiwe C, Lie AA, Beck H, Waha A, Friedl MG, Kuhn R, Emson P, Elger C, Wiestler OD. Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients. J Neuropathol Exp Neurol. 2000; 59: 1–10.[Medline] [Order article via Infotrieve]
  27. Sommer C, Roth SU, Kiessling M. Kainate induced epilepsy alters protein expression of AMPA receptor subunits GluR1, GluR2 and AMPA receptor binding protein (ABP) in the rat hippocampus. Acta Neuropathol (Berl). 2001; 101: 460–468.[Medline] [Order article via Infotrieve]
  28. Petralia RS, Wenthold RJ. Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain. J Comp Neurol. 1992; 318: 329–354.[CrossRef][Medline] [Order article via Infotrieve]
  29. Petralia RS, Wang Y-X, Mayat E, Wenthold RJ. Glutamate receptor subunit 2-selective antibody shows a differential distribution of calcium-impermeable AMPA receptors among populations of neurons. J Comp Neurol. 1997; 385: 456–476.[CrossRef][Medline] [Order article via Infotrieve]
  30. Wenthold RJ, Yokotani N, Doi K, Wada K. Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies: evidence for a hetero-oligomeric structure in rat brain. J Biol Chem. 1992; 267: 501–507.[Abstract/Free Full Text]
  31. Roth SU, Sommer C, Mundel P, Kiessling M. Expression of synaptopodin, a novel actin-associated protein, in the rat hippocampus after limbic epilepsy. Brain Pathol. 2001; 11: 169–181.[Medline] [Order article via Infotrieve]
  32. Pollard H, Héron A, Moreau J, Ben-Ari Y, Khrestchatisky M. Alterations of the GluR-B AMPA receptor subunit flip/flop expression in kainate-induced epilepsy and ischemia. Neuroscience. 1993; 57: 545–554.[CrossRef][Medline] [Order article via Infotrieve]
  33. Heurteaux C, Lauritzen I, Widmann C, Lazdunski M. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning. Proc Natl Acad Sci U S A. 1995; 92: 4666–4670.[Abstract/Free Full Text]
  34. Frank L, Diemer NH, Kaiser F, Sheardown M, Rasmussen JS, Kristensen P. Unchanged balance between levels of mRNA encoding AMPA glutamate receptor subtypes following global cerebral ischemia in the rat. Acta Neurol Scand. 1995; 92: 337–343.[Medline] [Order article via Infotrieve]
  35. Alsbo CW, Wrang ML, Johansen FF, Diemer NH. Quantitative PCR analysis of AMPA receptor composition in two paradigms of global ischemia. Neuroreport. 2000; 11: 311–315.[Medline] [Order article via Infotrieve]
  36. Friedman LK, Ginsberg MD, Belayev L, Busto R, Alonso OF, Lin B, Globus MY-T. Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia. Mol Brain Res. 2001; 86: 34–47.[Medline] [Order article via Infotrieve]
  37. Dienel GA, Pulsinelli WA, Duffy TE. Regional protein synthesis in rat brain following acute hemispheric ischemia. J Neurochem. 1980; 35: 1216–1226.[CrossRef][Medline] [Order article via Infotrieve]
  38. Thilmann R, Xie Y, Kleihues P, Kiessling M. Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus. Acta Neuropathol (Berl). 1986; 71: 88–93.[CrossRef][Medline] [Order article via Infotrieve]
  39. Nowa TS Jr, Kiessling M. Reprogramming of gene expression.In: Waltz W, ed. Cerebral Ischemia: Molecular and Cellular Physiology. Totowa, NJ: Humana Press; 1999: 145–215.
  40. Harrub JB, Nowak TS Jr. Cryptic expression of the 70-kDa heat shock protein, hsp72, in gerbil hippocampus after transient ischemia. Neurochem Res. 1998; 23: 703–708.[CrossRef][Medline] [Order article via Infotrieve]
  41. Optiz T, Grooms SY, Bennett MVL, Zukin RS. Remodeling of {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia. Proc Natl Acad Sci U S A. 2000; 97: 13360–13365.[Abstract/Free Full Text]
  42. Hatakeyama T, Matsumoto M, Brengman JM, Yanagihara T. Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils. Stroke. 1988; 19: 1526–1534.[Abstract/Free Full Text]
  43. Iwai T, Hara A, Niwa M, Nozaki M, Uematsu T, Sakai N, Yamada H. Temporal profile of nuclear DNA fragmentation in situ in gerbil hippocampus following transient forebrain ischemia. Brain Res. 1995; 671: 305–308.[CrossRef][Medline] [Order article via Infotrieve]
  44. Oguro K, Oguro N, Kojima T, Grooms SY, Calderone A, Zheng X, Bennett MVL, Zukin RS. Knockdown of AMPA receptor GluR2 expression causes delayed neurodegeneration and increases damage by sublethal ischemia in hippocampal CA1 and CA3 neurons. J Neurosci. 1999; 19: 9218–9227.[Abstract/Free Full Text]
  45. Kjøller C, Diemer NH. GluR2 protein synthesis and metabolism in rat hippocampus following transient ischemia and ischemic tolerance induction. Neurochem Int. 2000; 37: 7–15.[CrossRef][Medline] [Order article via Infotrieve]
  46. Jia Z, Agopyan N, Miu P, Xiong Z, Henderson J, Gerlai R, Taverna FA, Velumian A, MacDonald J, Carlen P, Abramow-Newerly W, Rode J. Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron. 1996; 17: 945–956.[CrossRef][Medline] [Order article via Infotrieve]
  47. Kask K, Zamanillo D, Rozov A, Burnashev N, Sprengel R, Seeburg PH. The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function. Proc Natl Acad Sci U S A. 1998; 95: 13777–13782.[Abstract/Free Full Text]
  48. Feldmeyer D, Kask K, Brusa R, Kornau H-C, Kolhekar R, Rozov A, Burnashev N, Jensen V, Hvalby Ø, Sprengel R, Seeburg PH. Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B. Nat Neurosci. 1999; 2: 57–64.[CrossRef][Medline] [Order article via Infotrieve]
  49. Bochet P, Audinat E, Lambolez B, Crepel F, Rossier J, Iino M, Tsuzuki K, Ozawa S. Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel. Neuron. 1994; 12: 383–388.[CrossRef][Medline] [Order article via Infotrieve]
  50. Geiger JR, Melcher T, Koh DS, Sakmann B, Seeburg PH, Jonas P, Monyer H. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron. 1995; 15: 193–204.[CrossRef][Medline] [Order article via Infotrieve]
  51. Tóth K, McBain CJ. Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons. Nat Neurosci. 1998; 1: 572–578.[CrossRef][Medline] [Order article via Infotrieve]
  52. Hsu M, Buzsáki G. Vulnerability of mossy fiber targets in the rat hippocampus to forebrain ischemia. J Neurosci. 1993; 13: 3964–3979.[Abstract]
  53. Maglóczky Z, Freund TF. Delayed neuronal death in the contralateral hippocampus following kainate injection into the CA3 subfield. Neuroscience. 1995; 66: 847–860.[CrossRef][Medline] [Order article via Infotrieve]
  54. Paschen W, Doutheil J, Uto A, Gissel C. Changes in endoplasmic reticulum Ca2+-ATPase mRNA levels in transient cerebral ischemia of rat: a quantitative polymerase chain reaction study. Neurosci Lett. 1996; 217: 41–44.[Medline] [Order article via Infotrieve]
  55. Stout AK, Raphael HM, Kanterewicz BI, Klann E, Reynolds IJ. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat Neurosci. 1998; 1: 366–373.[CrossRef][Medline] [Order article via Infotrieve]
  56. Mattson MP, LaFerla FM, Chan SL, Leissring MA, Shepel PN, Geiger JD. Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders. Trends Neurosci. 2000; 23: 222–229.[CrossRef][Medline] [Order article via Infotrieve]
  57. Alsbo CW, Wrang ML, Nielsen M, Diemer NH. Ischemic tolerance affects the adenylation state of GluR2 mRNA. Neuroreport. 2000; 11: 3279–3282.[Medline] [Order article via Infotrieve]
  58. Fan D, Grooms SY, Araneda RC, Johnson AB, Dobrenis K, Kessler JA, Zukin RS. AMPA receptor protein expression and function in astrocytes cultured from the hippocampus. J Neurosci Res. 1999; 57: 557–571.[CrossRef][Medline] [Order article via Infotrieve]
  59. Dong H, O’Brien RJ, Fung ET, Lanahan AA, Worley PF, Huganir RL. GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature. 1997; 386: 279–284.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
T. P. Obrenovitch
Molecular Physiology of Preconditioning-Induced Brain Tolerance to Ischemia
Physiol Rev, January 1, 2008; 88(1): 211 - 247.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
W.-R. Schabitz, C. Berger, R. Kollmar, M. Seitz, E. Tanay, M. Kiessling, S. Schwab, and C. Sommer
Effect of Brain-Derived Neurotrophic Factor Treatment and Forced Arm Use on Functional Motor Recovery After Small Cortical Ischemia
Stroke, April 1, 2004; 35(4): 992 - 997.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
C. Sommer, A. Fahrner, and M. Kiessling
[3H]Muscimol Binding to {gamma}-Aminobutyric AcidA Receptors Is Upregulated in CA1 Neurons of the Gerbil Hippocampus in the Ischemia-Tolerant State
Stroke, June 1, 2002; 33(6): 1698 - 1705.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sommer, C.
Right arrow Articles by Kiessling, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sommer, C.
Right arrow Articles by Kiessling, M.
Related Collections
Right arrow Gene expression
Right arrow Gene regulation
Right arrow Genomics
Right arrow Ischemic biology - basic studies

Stroke Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2002 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.